Methionine enkephalin (MENK) inhibits tumor growth through regulating CD4+Foxp3+ regulatory T cells (Tregs) in mice

Cancer Biol Ther. 2015;16(3):450-9. doi: 10.1080/15384047.2014.1003006.


Methionine enkephalin (MENK), an endogenous neuropeptide, plays an crucial role in both neuroendocrine and immune systems. CD4+Foxp3+ regulatory T cells (Tregs) are identified as a major subpopulation of T lymphocytes in suppressing immune system to keep balanced immunity. The aim of this research work was to elucidate the mechanisms via which MENK interacts with Tregs in cancer situation. The influence of MENK on transforming growth factor-β (TGF-β) mediated conversion from naïve CD4+CD25- T cells to CD4+CD25+ Tregs was determined and the data from flow cytometry (FCM) analysis indicated that MENK effectively inhibited the expression of Foxp3 during the process of TGF-βinduction. Furthermore, this inhibiting process was accompanied by diminishing phosphorylation and nuclear translocation of Smad2/3, confirmed by western blot (WB) analysis and immunofluorescence (IF) at molecular level. We established sarcoma mice model with S180 to investigate whether MENK could modulate Tregs in tumor circumstance. Our findings showed that MENK delayed the development of tumor in S180 tumor bearing mice and down-regulated level of Tregs. Together, these novel findings reached a conclusion that MENK could inhibit Tregs activity directly and retard tumor development through down-regulating Tregs in mice. This work advances the deepening understanding of the influence of MENK on Tregs in cancer situation, and relation of MENK with immune system, supporting the implication of MENK as a new strategy for cancer immunotherapy.

Keywords: CCR4, CC chemokine receptor 4; CTLA-4, cytotoxic T Lymphocyte Antigen-4; FCM, flow cytometry; FasL, Fas ligand; Foxp3, forkhead box P3 transcription factor; GITR, glucocorticoid-induced TNFR; IF, immunofluorescence; IL-2, interleukin-2; MENK, methionine enkephalin; MFI, median fluorescence intensity; MTS, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; Smad2/3 protein; TGF-β,transforming growth factor-β; Tregs, regulatory T cells; WB, protein gel blot; cancer immunoregulation; forkhead box P3 transcription factor; methionine enkephalin; real-time PCR, real-time polymerase chain reaction; regulatory T cell reprogramming; regulatory T cells; transforming growth factor-β.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CD4-Positive T-Lymphocytes / drug effects
  • CD4-Positive T-Lymphocytes / metabolism
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Enkephalin, Methionine / administration & dosage*
  • Flow Cytometry
  • Forkhead Transcription Factors / immunology
  • Forkhead Transcription Factors / metabolism*
  • Humans
  • Immunotherapy*
  • Mice
  • Sarcoma / drug therapy*
  • Sarcoma / metabolism
  • Sarcoma / pathology
  • T-Lymphocytes, Regulatory / drug effects
  • T-Lymphocytes, Regulatory / immunology
  • T-Lymphocytes, Regulatory / metabolism*
  • Transforming Growth Factor beta / immunology
  • Transforming Growth Factor beta / metabolism


  • Forkhead Transcription Factors
  • Foxp3 protein, mouse
  • Transforming Growth Factor beta
  • Enkephalin, Methionine